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Edizioni ETS Pisa, 2008
337
SHORT COMMUNICATION
THE SMALL GENOMIC SEGMENT OF TOMATO SPOTTED WILT VIRUS
ISOLATE BR20 IS NECESSARY BUT NOT SUFFICIENT
TO INDUCE LETHAL NECROSIS IN NICOTIANA BENTHAMIANA
AND LOCAL NECROTIC LESIONS IN NICOTIANA TABACUM cv. WHITE BURLEY
P. Margaria*, D. Pacifico*, M. Ciuffo and M. Turina
Istituto di Virologia Vegetale del CNR, Strada delle Cacce 73, 10135 Torino, Italy
SUMMARY
A consistently different phenotype was observed in
Nicotiana benthamiana and Nicotiana tabacum cv. White
Burley plants infected by the Brazilian Tomato spotted
wilt virus (TSWV) isolate Br20 and the two Italian isolates p272 and p105/43.14. Based on the phenotype of
experimentally obtained reassortants carrying the small
(S) genomic segment of p272 or p105/43.14 and the
medium (M) and large (L) segments of Br20, we concluded that the small segment of Br20 is required for eliciting
lethal necrosis in N. benthamiana and local necrotic lesions in N. tabacum. To evaluate whether the Br20 nucleocapsid (N) or the non structural (NSs) protein encoded
by the S segment were able to cause the distinctive symptoms, either alone or in combination, the three allelic
variants (Br20, p272 and p105/43.14) of each gene were
transiently expressed in planta through agroinfiltration.
All the alleles of the N and NSs proteins accumulated
abundantly in infiltrated N. benthamiana and N. tabacum
leaves. No specific necrotic reaction was observed in any
of the single or combined agro-infiltrations of the Br20
alleles, suggesting that the S-encoded proteins of isolate
Br20 are not sufficient to elicit the specific symptomatic
response and that the co-involvement of other regions of
the genome might be necessary.
Key words: Tospoviruses, Tomato spotted wilt virus,
symptom severity, reassortment, agroinfiltration.
Tomato spotted wilt virus (TSWV), the type member
of the genus Tospovirus, family Bunyaviridae, causes
major economic losses to a wide range of plants (German et al., 1992; Parrella et al., 2003). The viral genome
consists of three single-stranded RNA segments of negative or ambisense polarity. The large (L) segment (8.9
kb) encodes the RNA dependent RNA polymerase in
the complementary sense (de Haan et al., 1991). The
Corresponding author: M. Turina
Fax: +39.011.343809
E-mail: [email protected]
*
Both authors have contributed equally to this study
medium (M) segment (4.8 kb) encodes the glycoproteins precursor in the complementary sense and the
non-structural NSm protein in the viral sense (Kormenlink et al., 1992). The small (S) segment (2.9 kb) encodes the nucleocapsid (N) protein and the non-structural protein (NSs) in the complementary and viral
sense respectively (de Haan et al., 1990; Kormenlink et
al., 1991). In particular, the N protein is the main component of the viral nucleocapsid. Its involvement is crucial to the viral cycle, regulating functions in early
events of transcription and replication (Steinecke et al.,
1998). The same protein also interacts with the viral
polymerase, with the envelope glycoproteins, and with
the tubular structures formed by the NSm protein
(Uhrig et al., 1999). Moreover, it binds ssRNA in vitro
(Richmond et al., 1998) and interacts with the Gc protein in vivo (Snippe et al., 2007).
The NSs protein is expressed in infectious thrips
(Wijkamp et al., 1993) and accumulates in the salivary
glands, suggesting a possible role in vector transmission
(Goldbach et al., 1996). This protein has recently been
identified as a silencing suppressor (Bucher et al., 2003;
Takeda et al., 2002) and evidence for its involvement in
specific virus-host interactions has been given (Margaria
et al., 2007). The amount of NSs was hypothesized to be
associated with the severity of symptom expression (Kormenlink et al., 1991).
We observed a consistently different phenotype on
plants infected by the Brazilian TSWV isolate Br20 in
comparison with two previously characterized northern
Italian isolates, p272 and p105/43.14, capable of infecting Tsw resistant pepper (Margaria et al., 2007). Symptom differences were consistent in the environmental
conditions tested and previously described (Roggero et
al., 2002). However, inoculum obtained from the source
host Emilia sonchifolia prevented interference from defective RNAs (Inoue-Nagata et al., 1997). Br20 causes a
rapid lethal necrosis in N. benthamiana, about 10 days
post-inoculation, and local necrotic lesions in N.
tabacum cv. White Burley, in contrast to the Italian isolates, which both cause a mild mosaic without necrosis
and crumpling of the young leaves in N. benthamiana
and chlorotic symptoms in N. tabacum (Fig. 1 and data
not shown for isolate p272).
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Pathogenicity of Tomato spotted wilt virus BR20
In a previous work, reassortant isolates carrying the L
and M segments of Br20 and the S segment of p272 (reassortant isolate IFA201) or p105/43.14 (reassortant isolate
IFA231) were obtained (Margaria et al., 2007). Interestingly, these isolates elicited the mild symptoms typical of
the Italian parental strain in N. benthamiana and N.
tabacum, but no necrotic response (Fig. 1 and data not
shown for reassortant isolate IFA201). These findings
suggested that the S RNA was involved in determining
the differences in symptomatology, and that the S segment of Br20 was necessary for the induction of lethal
necrosis in N. benthamiana and local necrotic lesions in
N. tabacum.
Since the full S segment of the three parental isolates
had previously been sequenced (Margaria et al., 2007)
(Br20 accession No. DQ915948; p272 accession No.
DQ376181; p105/43.14 accession No. DQ376182), a
comparison of the Italian and Brazilian isolates showed
that several nucleotide (nt) mutations occurred in the
coding and non-coding regions of these sequences.
With respect to the proteins, one silent nt mutation was
observed between the two Italian isolates in the N gene.
Several nt and four amino acid (aa) differences were
present in the Br20 N protein-encoding ORF in comparison with the comparable Italian alleles. Moreover,
several mutations at the nt and aa level were always
present between the Italian and Brazilian NSs sequences. These differences allowed us to speculate on a
possible role of these proteins in eliciting the specific
Br20-associated necrotic response.
As a further step, we wanted to verify if a specific protein encoded by the Br20 S genomic segment was sufficient to elicit the specific necrotic symptoms. To this aim,
the entire S segments from the three virus isolates were
cloned in both orientations, and tested, either alone or in
combination, for their capability to induce the necrotic
Journal of Plant Pathology (2008), 90 (2), 337-343
response in agroinfiltrated plants. For cloning purposes,
viral nucleocapsids were purified from infected N. benthamiana leaves and nucleic acids extracted with phenolchloroform procedure and Na-Acetate/Ethanol precipitation (de Haan et al., 1990). The S RNA of each isolate
was purified from 1% TAE gel (Qbiogene, USA) and
used as a template to obtain cDNA using the J13 primer
containing the eight terminal nucleotides conserved in all
tospoviral RNA termini as described (Margaria et al.,
2007). A BamHI restriction site was inserted at the 5’ and
3’ ends to allow the ligation reaction of the purified PCR
product (Qiagen, USA) to the binary plasmid pBin61
(Bendahmane et al., 2002), in the presence of 3 unit of
the T4 DNA ligase (Promega, USA). Six recombinant
plasmids were obtained, carrying the S segment of Br20,
p272 or p105/43.14 in both orientations, to allow the expression of the two proteins encoded by each isolate under the 35S promoter and the Nos terminator (Tab. 1).
Each clone was sequenced to confirm that mutations
were not introduced into either of the two ORFs during
the amplification reaction by the thermo-stable DNA
polymerase (data not shown).
Liquid cultures of transformed C58C1 Agrobacterium
tumefaciens cells were used to infiltrate 15-20-day-old
plants of N. benthamiana and N. tabacum cv. White Burley (Bendahmane et al., 1999). To avoid experimental
variation, one half of each leaf was inoculated with the
Br20 derived construct and the other half with the analogous construct derived from an Italian isolate or with the
vector itself.
In order to have the simultaneous expression of the
two proteins encoded by the same isolate and check for
possible interactions between the two viral proteins that
could cause the induction of necrosis, a mixture was also infiltrated of two A. tumefaciens cultures transformed
with the two clones derived from the same S RNA in
Table 1. Constructs and derived recombinant proteins used in this study. v-mRNA= viral mRNA; vc-mRNA= viral complementary mRNA.
pBin61a derived Constructs
pBin61-Br20N
pBin61-Br20NSs
pBin61-p272N
pBin61-p272NSs
pBin61-p105/43.14N
pBin61-p105/43.14NSs
pBin61-TBSVep19
pBin61-GFPg
a
Encoded mRNA
TSWVb vc-mRNA
TSWV v-mRNA
TSWV vc-mRNA
TSWV v-mRNA
TSWV vc-mRNA
TSWV v-mRNA
p19f mRNA
GFP mRNA
pBin61 is the binary plasmid used for agroinfiltration Bendhamane et al., 2002
TSWV= Tomato spotted wilt virus
c
N= nucleocapsid protein from TSWV
d
NSs= non structural protein from S segment of TSWV
e
TBSV= Tomato bushy stunt virus
f
p19= protein from TBSV known to cause severe necrosis
g
GFP= green fluorescent protein
b
Encoded protein
Nc
NSsd
N
NSs
N
NSs
p19
GFP
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Margaria et al.
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Fig. 1. Comparison of the symptoms observed in plants infected by the isolates in consideration: p105/43.14 (left), Br20 (middle)
and the reassortant IFA231 (right), carrying the S segment of isolate p105/43.14 and the M and L segments of isolate Br20. A)
Photographs of N. benthamiana 10 days after inoculation showing lethal necrosis (Br20) or a mild mosaic without necrosis associated with crumpling of the young leaves (p105/43.14 and reassortant IFA231). B) Photographs of N. tabacum 7 days after inoculation showing local necrotic lesions (Br20) or chlorotic symptoms (p105/43.14 and reassortant IFA231).
both orientations (not shown).
As a positive control for a viral protein causing lethal
necrosis and local necrotic lesions, we agroinfiltrated
the pBin61 vector carrying the p19 gene of Tomato
bushy stunt virus (TBSV) (Scholthof et al., 1995). Vector
pBin61 alone or a pBin61-GFP vector expressing the
Green fluorescent protein (Margaria et al., 2007) served
as negative control.
Western blot analysis was used to assess the in planta
expression of each of the viral proteins encoded by the S
segment and symptoms were monitored to detect any specific phenotypic reaction associated with the expression of
a specific allele. Plants were kept under greenhouse conditions (Roggero et al., 2002) and the expression of proteins was examined after three days by Western blot
analysis using the primary antibodies A514 (Heinze et al.,
1998) and AE353 (Roggero et al., 1998) to detect the N
and NSs protein, respectively. Symptom development on
the agroinfiltrated plants was monitored daily.
All plants tested positive for protein expression by
Western blot analysis three days after agroinfiltration.
Both the N and the NSs proteins of Br20 and the Italian
isolates accumulated abundantly in the plant hosts after
single (Fig. 2 and data not shown for isolate p105/
43.14) or combined (not shown) infiltration with the respective clones. The Br20 N protein accumulated at a
lower level compared to the alleles of the Italian strains,
while no difference was observed in the accumulation
level of the NSs proteins.
No specific symptom development associated with
the expression of either of the two viral proteins,
whether alone or in combination, was observed (Fig. 3
and Table 2). Furthermore, there was no specific response in the negative control, except for chlorotic
spots induced on N. tabacum 7 days post inoculation.
As expected, we observed a strong necrotic reaction
when the pBin61 plasmid encoding the TBSV p19 protein was agroinfiltrated and a green fluorescence in the
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Pathogenicity of Tomato spotted wilt virus BR20
Journal of Plant Pathology (2008), 90 (2), 337-343
Table 2. Presence (+) or absence (-) of necrotic symptoms on N. benthamiana and N. tabacum cv. White Burley infiltrated
with different pBin61 constructs. Necrosis was observed only in plants expressing the TBSV p19 protein positive control.
Encoded
protein
N. benthamiana
N. tabacum
TBSVp19 GFP Br20
N
+
+
-
Br20
NSs
-
p272
N
-
p272
NSs
-
Fig. 2. Western immunoblot detection of the N (panel A) and
NSs (panel B) proteins from both Br20 and p272 three days
after infiltration in N. tabacum (Nt) and N. benthamiana (Nb).
Note the differences in the accumulation level of the N protein. The same situation was observed for the Br20 and
p105/43.14 proteins (not shown).
p105/43.14
N
-
p105/43.14
NSs
-
Br20
N+NSs
-
p272
N+NSs
-
p105/43.14
N+NSs
-
plants infiltrated with the pBin61-GFP construct (Fig. 3
and data not shown). We also attempted agroinfiltration
of isolate Br20 S-encoded protein in leaves infected
with the mild Italian strains, but no necrosis developed
(data not shown).
Several determinants have been associated with
virus-induced specific symptoms in host plants, i.e.
whole ORFs (de Assis Filho et al., 2002; Kagiwada et
al., 2005; Szilassi et al., 1999; Tribodet et al., 2005), non
coding sequences (Rodriguez-Cerezo et al., 1991; van
der Vossen et al., 1996; Zhang et al., 1994), or silent mutations (Hirata et al., 2003).
A limited number of studies have been published on
the biology of TSWV-host plant interaction (Best, 1961;
Hoffmann et al., 2001; Jahn et al., 2000; Mandal et al.,
2006; Margaria et al., 2007; Pang et al., 1993; Qiu et al.,
1998, 1999). Pseudoricombination was used to investigate the biological properties of the different segments
of the multipartite TSWV genome, by obtaining reassortants from parental strains with different biological
properties, and analyzing their symptom expression in
different host plants (Best, 1961; Pang et al., 1993; Qiu
et al., 1998). For instance, Qiu et al. (1998) showed that
the lesion diameter in Cucumis sativus cv. National Pickling maps to segment M and infection to N. tabacum cv.
White Burley to segment L of TSWV RNA. In similar
experiments, M and the S RNAs of the Hawaian isolate
TSWV-A proved to be associated with the ability to
overcome the resistance conferred by the Sw5 and Tsw
genes to tomato and pepper, respectively (Hoffmann et
al., 2001; Jahn et al., 2000). Influence of the intergenic
region (IGR) of segment S in the biological properties of
TSWV was studied by Qiu et al. (1998), who reported
an involvement of the IGR length in the competitiveness
of the reassortment process. Similar results are available
for the M segment (Hoffmann et al., 2001). Reassortment was also used by Okuda et al. (2003) to show that
the S segment of the taxonomically related Watermelon
silver mottle virus was associated with differences in the
symptom in Tetragonia expansa.
The influence of the S RNA segment on biological
properties was also determined for members of another
genus (Phlebovirus) in the family Bunyaviridae that infects animals and humans (Bridgen et al., 2001; Perrone
et al., 2007; Vialat et al., 2000).
Previous results have shown that the determinants for
biological properties and specific symptomatic expression in TSWV map to different genomic segments ac-
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Margaria et al.
A
B
C
D
341
Fig. 3. Symptoms induced by agroinfiltration of different pBin61 derived constructs on N. tabacum. No specific reaction was observed when the pBin61 vector encoding the N (A) or NSs (B) protein of Br20 or p272 was agroinfiltrated. The same situation
was observed for the p105/43.14 constructs (not shown). C) Necrotic reaction induced by the expression vector carrying the p19
protein gene of Tomato bushy stunt virus. D) Chlorotic spots induced by the pBin61 expression vector 7 days post infiltration.
cording to the host plant and the property considered.
However, very little is known about specific determinants
responsible for the induction of specific symptoms.
In this work, experimentally derived reassortants allowed us to prove that the S RNA of Br20 is necessary for
the necrotic response of N. benthamiana and N. tabacum
cv. White Burley. Using A. tumefaciens as a delivery and
expression system, we obtained the in planta transcription and expression of the alleles of both viral proteins
encoded by S RNA. Previous data showed transient heterologous expression to be a suitable tool to investigate
pathogen/host interactions and to study single gene effects on plant response without virus interference (Annamali et al., 2005; Marillonet et al., 2005; Scholthof et al.,
1996; Takeda et al., 2002; Voinnet et al., 2000). In addi-
tion, the transient approach is a quick way to achieve expression of recombinant proteins, while avoiding the
lethal necrosis induced by necrosis-inducing proteins in
the early stage of development of stable transgenic plants.
Co-agroinfiltration of different constructs is also a common tool used to study protein-protein interaction at the
single cell level, and a number of reports confirm the in
vivo delivery in the same cell of multiple constructs (Canto et al., 2007; Li and Chye, 2004).
No specific necrotic symptoms related to the N and
NSs viral proteins encoded by segment S of Br20 were
observed, indicating that they are not sufficient,
whether alone or in combination, for inducing the
necrotic response. Kormenlink et al. (1991) suggested
that a correlation exists between the amount of NSs and
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Pathogenicity of Tomato spotted wilt virus BR20
the severity of symptoms, for severe symptoms were
elicited by isolates expressing higher amounts of the
NSs protein than the mild strains. Our data detected no
difference in the level of accumulation of the NSs protein between Br20 and the TSWV Italian isolates. The
absence of symptom expression in the combined infiltrations seems also to exclude the possibility that the interaction between the two S RNA-encoded proteins induces the specific response. Moreover, also the corresponding mRNAs, either alone or in combination, are
not sufficient for necrotic symptoms development.
Therefore, we suggest that the non coding sequences
of S RNA or a synergic interaction between elements of
S RNA and elements of the M and/or L RNA may be
involved in the necrotic response. Previous works showing that the N protein interacts with proteins encoded
by other genomic segments (Snippe et al., 2007; Uhrig
et al., 1999) support this hypothesis. No interaction between the NSs protein and those encoded by other genomic segments has been reported so far, nor between
the proteins encoded by the S RNA and plant factors.
Lack of a reverse genetic system for TSWV hampers
the possibility to further explore the complexity of the
necrotic response in N. benthamiana and N. tabacum.
ACKNOWLEDGEMENTS
We thank Caterina Perrone for technical assistance
and Jeness Petersen for editing the manuscript.
REFERENCES
Annamali P., Rao A.L., 2005. Replication-independent expression of genome components and capsid protein of
brome mosaic virus in planta: a functional role for viral
replicase in RNA packaging. Virology 338: 96-111.
Bendahmane A., Kanyuka K., Baulcombe D.C., 1999. The Rx
gene from potato controls separate virus resistance and cell
death responses. Plant Cell 11: 781-792.
Bendahmane A., Farnham G., Moffett P., Baulcombe, D.C.,
2002. Constitutive gain-of-function mutants in a nucleotide binding site-leucine rich repeat protein encoded at
the Rx locus of potato. Plant Journal 32: 195-204.
Best R.J., 1961. Recombination experiments with strains A
and E of Tomato spotted wilt virus. Virology 15: 327-339.
Bridgen A., Weber F., Fazakerley J.K., Elliott R.M., 2001.
Bunyamwera bunyavirus nonstructural protein NSs is a
nonessential gene product that contributes to viral pathogenesis. Proceedings of the National Academy of Sciences
USA 98: 664-669.
Bucher E., Sijen T., De Haan P., Goldbach R., Prins M., 2003.
Negative-strand tospoviruses and tenuiviruses carry a gene
for a suppressor of gene silencing at analogous genomic
positions. Journal of Virology 77: 1329-1336.
Canto T., Uhrig J.F., Swanson M., Wright K.M., MacFarlane
S.A., 2006. Translocation of Tomato bushy stunt virus P19
Journal of Plant Pathology (2008), 90 (2), 337-343
protein into the nucleus by ALY proteins compromises its
silencing suppressor activity. Journal of Virology 80: 90649072.
de Assis Filho F.M., Paguio O.R., Sherwood J.L., Deom C.M.,
2002. Symptom induction by Cowpea chlorotic mottle virus
on Vigna unguiculata is determined by amino acid residue
151 in the coat protein. Journal of General Virology 83:
879-883.
de Haan P., Wagemakers L., Peters D., Goldbach R., 1990.
The S RNA segment of tomato spotted wilt virus has an
ambisense character. Journal of General Virology 71: 10011007.
de Haan P., Kormelink R., de Oliveira Resende R., van Poelwijk F., Peters D., Goldbach, R., 1991. Tomato spotted wilt
virus L RNA encodes a putative RNA polymerase. Journal
of General Virology 72: 2207-2216.
German T.L., Ullman D.E., Moyer J.W., 1992. Tospoviruses:
diagnosis, molecular biology, phylogeny and vector relationship. Annual Review of Phytopathology 30: 315-348.
Goldbach R., Peters D., 1996. Molecular and biological aspects of Tospoviruses In: The Bunyaviridae. Elliot R.M.
(ed), pp. 129-157. Plenum Press, New York, NY, USA.
Heinze C., Roggero P., Sohn M., Ciuffo M., Masenga V., Vaira
A.M., Adam, G., 1998. Antisera against a conserved domain of Tospovirus NSs-protein: characterization and epitope mapping. Proceedings of the 4th International Symposium on tospoviruses and thrips in floral and vegetable crops,
Wageningen 1988: 29-30.
Hirata H., Lu X., Yamaji Y., Kagiwada S., Ugaki M., Namba
S., 2003. A single silent substitution in the genome of Apple stem grooving virus causes symptom attenuation. Journal of General Virology 84: 2579-2583.
Hoffmann K., Qiu W.P., Moyer J.W., 2001. Overcoming hostand pathogen-mediated resistance in tomato and tobacco
maps to the M RNA of Tomato spotted wilt virus. Molecular Plant-Microbe Interaction 14: 242-249.
Inoue-Nagata A.K., Kormelink R., Nagata T., Kitajima E.W.,
Goldbach R., Peters D., 1997. Effects of temperature and
host on the generation of tomato spotted wilt virus defective interfering RNAs. Phytopathology 87: 1168-1173.
Jahn M., Paran I., Hoffmann K., Radwanski E.R., Livingstone
K.D., Grube R.C., Aftergoot E., Lapidot M., Moyer J.,
2000. Genetic mapping of the Tsw locus for resistance to
the tospovirus Tomato spotted wilt virus in Capsicum spp.
and its relationship to the Sw-5 gene for resistance to the
same pathogen in tomato. Molecular Plant-Microbe Interaction 13: 673-682.
Kagiwada S., Yamaji Y., Komatsu K., Takahashi S., Mori T.,
Hirata H., Suzuki M., Ugaki M., Namba S., 2005. A single
amino acid residue of RNA-dependent RNA polymerase in
the Potato virus X genome determines the symptoms in
Nicotiana plants. Virus Research 110: 177-182.
Kormelink R., Kitajima E.W., De Haan P., Zuidema D., Peters
D., Goldbach R., 1991. The nonstructural protein NSs encoded by the ambisense S RNA segment of tomato spotted
wilt virus is associated with fibrous structures in infected
plant cells. Virology 181: 459-468.
Kormelink R., de Haan P., Meurs C., Peters D., Goldbach R.,
1992. The nucleotide sequence of the M RNA segment of
tomato spotted wilt virus, a bunyavirus with two am-
021_JPP_136SC_337_colore
21-07-2008
12:00
Pagina 343
Journal of Plant Pathology (2008), 90 (2), 337-343
bisense RNA segments. Journal of General Virology 73:
2795-2804.
Li H.Y., Chye, M.L., 2004. Arabidopsis acyl-CoA-binding
protein ACBP2 interacts with an ethylene-responsive element-binding protein, AtEBP, via its ankyrin repeats. Plant
Molecular Biology 54: 233-243.
Mandal B., Pappu H.R., Csinos A.S., Culbreath A.K., 2006.
Response of peanut, pepper, tobacco, and tomato cultivars
to two biologically distinct isolates of Tomato spotted wilt
virus. Plant Disease 90: 1150-1155.
Margaria P., Ciuffo M., Pacifico D., Turina M., 2007. Evidence that the NSs protein of Tomato spotted wilt virus is
the avirulence determinant in the interaction with resistant
pepper carrying the Tsw gene. Molecular Plant-Microbe Interaction 20: 547-558
Marillonet S., Thoeringer C., Kandzia R., Klimyuk V., Gleba
Y., 2005. Systemic Agrobacterium tumefaciens-mediated
trasfection of viral replicons for efficient transient expression in plants. Nature Biotechnology 23: 718-723.
Okuda M., Taba S., Hanada K., 2003. The S RNA segment
determines symptom differences on Tetragonia expansa between two Watermelon silver mottle virus isolates. Physiological and Molecular Plant Pathology 62: 327-332.
Pang S.Z., Slightom J.L., Gonsalves D., 1993. The biological
properties of a distinct tospovirus and sequence analysis of
its RNA. Phytopathology 83: 728-733.
Parrella G., Gognalons P., Gebre-Selassié K., Vovlas C., Marchoux G., 2003. An update of the host range of Tomato
spotted wilt virus. Journal of Plant Pathology 85: 227-264.
Perrone L.A., Narayanan K., Worthy M., Peters C.J., 2007.
The S segment of Punta Toro virus (Bunyaviridae, Phlebovirus) is a major determinant of lethality in the Syrian
hamster and codes for a type I interferon antagonist. Journal of Virology 81: 884-892.
Qiu W.P., Geske S.M., Hickey C.M., Moyer J.W., 1998.
Tomato spotted wilt tospovirus genome reassortment and
genome segment-specific adaptation. Virology 244: 186194.
Qiu W.P., Moyer J.W., 1999. Tomato spotted wilt tospovirus
adapts to the TSWV derived resistance by genome reassortment. Phytopathology 89: 575-582.
Richmond K.E., Chenault K., Sherwood J.L., German T.L.,
1998. Characterization of the nucleic acid binding properties of tomato spotted wilt virus nucleocapsid protein. Virology 248: 6-11.
Rodriguez-Cerezo E., Klein P.G., Shaw J.G., 1991. A determinant of disease symptom severity is located in the 3’-terminal noncoding region of the RNA of a plant virus. Proceedings of the National Academy of Sciences USA 88: 9863-9867.
Roggero P., Ciuffo M., Vaira A.M., Milne R.G., 1998. Rapid
purification of tospovirus nucleocapsids for antibody production and RNA analysis. Proceedings of the 4th International Symposium on tospoviruses and thrips in floral and
vegetable crops, Wageningen 1998: 25-28.
Roggero P., Masenga V., Tavella L., 2002. Field isolates of
Tomato spotted wilt virus overcoming resistance in pepper
and their spread to other hosts in Italy. Plant Disease 86:
950-954.
Received November 29, 2007
Accepted February 12, 2008
Margaria et al.
343
Scholthof H.B., Scholthof K.G.B., Jackson A.O., 1995. Identification of Tomato bushy stunt virus host-specific symptom determinants by expression of individual genes from a
Potato virus X vector. Plant Cell 7: 1157-1172.
Scholthof H.B., Scholthof K.G.B., Jackson A.O., 1996. Plant
virus gene vectors for transient expression of foreign proteins in plants. Annual Review of Phytopathology 34: 299323.
Snippe M., Willem Borst J., Goldbach R., Kormelink R.,
2007. Tomato spotted wilt virus Gc and N proteins interact in vivo. Virology 357: 115-123.
Steinecke P., Heinze C., Oehmen E., Adam G., Schreier P.H.,
1998. Early events of tomato spotted wilt transcription and
replication in protoplasts. New Microbiology 21: 263-268.
Szilassy D., Salánki K., Balázs E., 1999. Stunting induced by
cucumber mosaic cucumovirus-infected Nicotiana glutinosa is determined by a single amino acid residue in the
coat protein. Molecular Plant-Microbe Interaction 12: 11051113.
Takeda A., Sugiyama K., Nagano H., Mori M., Kaido M.,
Mise K., Tsuda S., Okuno T., 2002. Identification of a novel RNA silencing suppressor, NSs protein of Tomato spotted wilt virus. FEBS Letters 532: 75-79.
Thomas-Carroll M.L., Jones R.A.C., 2003. Selection, biological properties and fitness of resistance-breaking strains of
Tomato spotted wilt virus in pepper. Annals of Applied Biology 142: 235-243.
Tribodet M., Glais L., Kerlan C., Jacquot E., 2005. Characterization of Potato virus Y molecular determinants involved
in the vein necrosis symptom induced by PVYN isolates in
infected Nicotiana tabacum cv. Xanthi. Journal of General
Virology 86: 2101-210.
Uhrig J.F., Soellick T.R., Minke C.J., Philipp C., Kellmann
J.W., Schreier P.H., 1999. Homotypic interaction and multimerization of nucleocapsid protein of tomato spotted wilt
tospovirus: identification and characterization of two interacting domains. Proceedings of the National Academy of
Sciences USA 96: 55-60.
van der Vossen E.A.G., Neeleman L., Bol J.F., 1996. The 5’
terminal sequence of alfalfa mosaic virus RNA3 is dispensable for replication and contains a determinant for symptom formation. Virology 221: 271-280.
Vialat P., Billecocq A., Kohl A., Bouloy M., 2000. The S segment of Rift Valley fever phlebovirus (Bunyaviridae) carries determinants for attenuation and virulence in mice.
Journal of Virology 74: 1538-1534.
Voinnet O., Rivas S., Mestre P., Baulcombe D.C., 2003. An
enhanced transient expression system in plants based on
suppression of gene silencing by the p19 protein of tomato
bushy stunt virus. The Plant Journal 33: 949-956.
Wijkamp I., van Lent J., Kormenlink R., Goldbach R., Peters
D., 1993. Multiplication of Tomato spotted wilt virus in its
vector Frankliniella occidentalis. Journal of General Virology 74: 341-349.
Zhang L., Hanada K., Palukaitis P., 1994. Mapping local and
systemic symptom determinants of cucumber mosaic cucumovirus in tobacco. Journal of General Virology 75: 31853191.
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